SUMMARY Although disruption of 3D genome organization is connected to human diseases, including developmental syndromes and cancer, there remain fundamental gaps in our understanding of how specific molecular machines control genome organization. Our long-term goal is to determine how evolutionarily conserved molecular mechanisms connect 3D genome organization and transcriptional regulation. We have recently discovered molecular links between a key genome organizer, condensin, and the transcriptional machinery (Mol. Cell 2015; Nature Genet. 2016). The objective here is to delineate how condensin connects 3D genome organization, chromosomal segregation, and transcriptional regulation. The central hypothesis is that the condensin interactions with TBP, mediator, TFIIIC and transcription factors (TFs) are required for associations among highly transcribed (HT) genes and centromeres, which promotes faithful segregation of chromosomes during mitosis, and that condensin controls gene transcription via 3D genome organization (gene positioning at centromeres) or/and local chromosomal compaction. We will test our hypothesis in the highly tractable fission yeast model (Aims 1 & 2) and characterize the more complex mechanisms in human cells using knowledge from the yeast system (Aim 3). Under Aim 1, we will test the hypothesis that the association between HT gene loci and centromeres, mediated by condensin-TBP interaction, is a stable structure to mediate proper chromosomal segregation (live-cell imaging with wt and condensin mutant). We will determine how merely positioning a gene locus at the centromere impairs transcription (lacO-based tethering). We will also test an alternative, local mechanism, whereby condensin represses transcription of HT genes (qRT-PCR) via DNA renaturation (nuclease assay) followed by local compaction (FISH). Under Aim 2, we will establish how transcription-related factors (TBP, mediator, TFIIIC & TFs) contribute to condensin loading and gene positioning at centromeres (tethering system & FISH). We will elucidate how the condensin-mediator interaction participates in chromosomal segregation and transcriptional regulation (FISH, RNA-seq & qRT-PCR with condensin mutant). Moreover, we will determine how the condensin interactions with TBP, mediator and TFs organize genome-wide associations and domains (ChIA-PET & Hi-C with condensin and TF mutants). Under Aim 3, we will use techniques from Aims 1 and 2 and condensin mutations in human RPE1 cells (in hand), and characterize how the interaction between condensin I and hTBP is involved in (1) removal of RNA polymerases from HT genes during mitosis and gene bookmarking; and (2) associations of HT genes and domain formation. The innovation of this project resides in our new concept connecting condensin-mediated 3D genome organization, chromosomal segregation, and transcriptional regulation. The proposed research is significant, because it is expected to improve the understanding of how condensin establishes the functional chromosomal structure, which remains much less understood than the cohesin mechanisms.